CNS regeneration is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. Inhibition of ROCK has been previously shown to promote regeneration in retinal ganglion cells (RGC) whereas Cdk5 inhibition mainly promoted survival. Therefore, we have evaluated the effects of combined treatment with inhibitors of ROCK and Cdk5. We show that in vitro, the co-application of the Cdk5 inhibitor, Indolinone A, and the ROCK inhibitor, Y-27632, potentiated the survival-promoting effect of either substance alone. However, neurite outgrowth in vitro was promoted only by the presence of Y-27632, not by Indolinone A alone. In the ex vivo explant and the in vivo optic nerve crush model the combination of both inhibitors significantly increased neurite outgrowth at small distances, but this effect leveled off for longer neurites. In Summary, the combined treatment with the Cdk5 inhibitor Indolinone A and the ROCK inhibitor Y-27632 results in a strong additive effect on neuronal survival, but is not able to increase the regenerative response beyond the effect of the ROCK inhibitor. (C) 2009 Elsevier Inc. All rights reserved.